Thermally responsive cell culture surfaces

ABSTRACT

A stimuli responsive nanofiber that includes a stimuli responsive polymer, such as a thermally responsive polymer, and a cross-linking agent having at least two latent reactive activatable groups. The nanofiber may also include a biologically active material or a functional polymer. The stimuli responsive nanofiber can be used to modify the surface of a substrate. When the nanofiber includes a thermally responsive polymer, the physical properties of the surface can be controlled by controlling the temperature of the system, thus controlling the ability of the surface to bind to a biologically active material of interest.

TECHNICAL FIELD

The present invention generally relates to stimuli responsive nanofibersand stimuli responsive nanofiber modified surfaces. More particularly,the present inven is directed to nanofibers including a thermallyresponsive polymer, a multi-functiona cross-linking agent, andoptionally a biologically active material or a functional poly that isreactive with a biologically active material. The stimuli responsivenanofibers be used to modify a surface of a substrate, such as a cellculture device.

BACKGROUND

Nanofibers are being considered for a variety of applications because oftheir unique properties including high surface area, small fiberdiameter, layer thinness, hig permeability, and low basis weight. Moreattention has been focused on functionaliz nanofibers having thecapability of incorporating active chemistry, especially in biomedicalapplications such as wound dressing, biosensors and scaffolds for tissueengineering.

Nanofibers may be fabricated by electrostatic spinning (also referred toas electrospinning). The technique of electrospinning of liquids and/orsolutions capab forming fibers, is well known and has been described ina number of patents, such as example, U.S. Pat. Nos. 4,043,331 and5,522,879. The process of electrospinning generally involves theintroduction of a solution or liquid into an electric field, so thasolution or liquid is caused to produce fibers. These fibers aregenerally drawn to a conductor at an attractive electrical potential forcollection. During the conversion o solution or liquid into fibers, thefibers harden and/or dry. This hardening and/or dry may be caused bycooling of the liquid, i.e., where the liquid is normally a solid at rtemperature; by evaporation of a solvent, e.g., by dehydration(physically induced hardening); or by a curing mechanism (chemicallyinduced, hardening).

The process of electrostatic spinning has typically been directed towardthe u the fibers to create a mat or other non-woven material, asdisclosed, for example, in Pat. No. 4,043,331. Nanofibers ranging from50 nm to 5 μm in diameter can be electrospun into a nonwoven or analigned nanofiber mesh. Due to the small fiber diameters, electrospuntextiles inherently possess a very high surface area and a small poresize. These properties make electrospun fabrics potential candidates fora number of applications including: membranes, tissue scaffolding, andother biomedical applications

Nanofibers can be used to modify the surface of a substrate to achieve adesired surface characteristic. Most nanofiber surfaces have to beengineered to obtain the ability to immobilize biomolecules. Surfacemodification of synthetic biomaterials, with the intent to improvebiocompatibility, has been extensively studied, and many commontechniques have been considered for polymer nanofiber modification. Forexample, Sanders et al in “Fibro-Porous Meshes Made from PolyurethaneMicro-Fibers: Effects of Surface Charge on Tissue Response” Biomaterials26, 813-818 (2005) introduced different surface charges on electrospunpolyurethane (PU) fiber surfaces through plasma-induced surfacepolymerization of negatively or positively charged monomers. The surfacecharged PU fiber mesh was implanted in rat subcutaneous dorsum for 5weeks to evaluate tissue compatibility, and it was found that negativelycharged surfaces may facilitate vessel ingrowth into the fibroporousmesh biomaterials. Ma et al. in “Surface Engineering of ElectrospunPolyethylene Terephthalate (PET) Nanofibers Towards Development of a NewMaterial for Blood Vessel Engineering” Biomaterials 26, 2527-2536 (2005)conjugated gelatin onto formaldehyde pretreated polyethyleneterephthalate (PET) nanofibers through a grafted polymethacrylic acidspacer and found that the gelatin modification improved the spreadingand proliferation of endothelial cells (ECs) on the PET nanofibers, andalso preserved the EC's phenotype. Chua et al. in “Stable Immobilizationof Rat Hepatocyte Spheroids on Galactosylated Nanofiber Scaffold”Biomaterials 26, 2537-2547 (2005) introduced galactose ligand ontopoly(e-caprolactone-co-ethyl ethylene phosphate) (PCLEEP) nanofiberscaffold via covalent conjugation to a poly(acrylic acid) spacerUV-grafted onto the fiber surface. Hepatocyte attachment, ammoniametabolism, albumin secretion and cytochrome P450 enzymatic activitywere investigated on the 3-D galactosylated PCLEEP nanofiber scaffold aswell as the functional 2-D film substrate

SUMMARY

The methods and techniques summarized above are costly, complicated, ormaterial specific. Thus, there is a need for a surface modificationapproach that is more general and easy to use and can be applied underMild conditions to a wide variety of nanofibers.

According to one embodiment, the present invention is a stimuliresponsive nanofiber including a stimuli responsive polymer. One exampleof a stimuli responsive nanofiber is a thermally responsive nanofiberincluding a thermally responsive polymer. In either of theseembodiments, the stimuli responsive nanofiber may include across-linking agent having at least two latent reactive activatablegroups. In use, photochemically, electrochemically or thermally latentreactive groups will form convalent bonds when subjected to a source ofenergy. Suitable energy sources include radiation electrical and thermalenergy. In some embodiments, the radiation energy is visible,ultraviolet, infrared, x-ray or microwave electromagnetic radiation.

The cross-linking agent may have at least two latent reactiveactivatable groups. These latent reactive groups may be the same or maybe different. For example, all of the latent reactive groups may bephotochemically reactive groups. Alternatively, in other embodiments ofthe invention the cross-linking agent may include both photochemicallyand thermally reactive groups. Further, the cross-linking agent may bemonomeric or polymeric materials or may be a mixture of both monomericand polymeric materials.

According to a further embodiment of the present invention, thethermally responsive polymer is poly(isopropylacrylamide) as well asderivatives of poly(isopropylacrylamide) such as graft copolymerderivatives with polyethylene glycol derivatives.

According to another embodiment, the present invention is a method oftreating a surface of a substrate including the steps of combining astimuli responsive polymer, such as a thermally responsive polymer, anda cross-linking agent having at least two latent reactive activatablegroups; forming at least one nanofiber from the combined mixture;contacting the surface with the nanofiber; and forming a bond betweenthe nanofiber and the surface.

According to another embodiment, the present invention is as surfacecoating for a surface of an article. The surface coating includes astimuli responsive nanofiber including a nanofiber coated with a stimuliresponsive polymer, such as a thermally responsive polymer, and across-linking agent having at least two latent reactive activatablegroups. Optionally, the coated nanofiber or the coated surface mayinclude a biologically active material or, alternatively, a functionalpolymer.

According to yet another embodiment, the present invention is an articleincluding a surface coating, having a thermally responsive nanofiber.According to a further embodiment, the thermally responsive nanofiberincludes a thermally responsive polmer and a cross-linking agent havingat least two latent reactive activatable groups.

According to still yet another embodiment, the present invention is acell culture device including a surface coating having a thermallyresponsive nanofiber. The thermally responsive nanofiber includes athermally responsive polymer, a cross-linking agent having at least twolatent reactive activatable groups, and a biologically active material.

According to other embodiments of the present invention, the stimuliresponsive nanofiber may have a diameter ranging from 1 to 100 micronsand still other embodiments may have a diameter ranging from 1 am to1000 nm. The stimuli responsive nanofiber may have an aspect ratio in arange of about at least 10 to at least 100.

According to yet a further embodiment of the present invention, thethermally responsive nanofiber has first physical property at a firstpredetermined temperature range and a second physical property at asecond predetermined temperature range. The thermally responsivenanofiber is capable of transitioning from a first physical property toa second physical property upon the application or removal of heat to orfrom the system

Various modifications and additions can be made to the exemplaryembodiments discussed without departing from the scope of the presentinvention. For example, while the embodiments described above refer toparticular features, the scope of this invention also includesembodiments having different combinations of features and embodimentsthat do not include all of the described features. Accordingly, thescope of the present invention is intended to embrace all suchalternatives, modifications, and variations as fall within the scope ofthe claims, together with all equivalents thereof.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is an electronic image of to polycaprolactone nanofiber describedin Example 1.

FIG. 2 is an electronic image of a polyisopropylacrylamide nanofiberdescribed in Example 3.

FIGS. 3 and 4 illustrate protein absorption profiles ofpolyisopropylacrylamide coated polystyrene and polyisopropylacrylamidenanofibers described in Example 4.

FIGS. 5 and 6 are electronic images of cell lift up times from varioussurfaces described in Example 5.

FIGS. 7A through 7D include electronic images of T47-D cells cultured onnanofiber and flat surfaces. FIG. 7A shows multicellular spheroids oncoated nanofibers. FIG. 7B is a magnified image of a spheroid shown inFIG. 7A. FIG. 7C shows spread out cellular morphology on coated TCPS.FIG. 7D shows cells lifted off and replated on nanofibers maintainmorphology and peripheral organization.

FIG. 8 is an electronic image of replated BAEC cells.

DETAILED DESCRIPTION

Stimuli responsive or “smart” materials are materials that have one ormore properties that can be altered in a controlled fashion by theapplication of external stimuli, such as stress, temperature, moisture,pH, applied electric or magnetic fields, ionic strength, or biomoleculessuch as glucose or antigens. Representative stimuli responsive materialsand polymers as well as their physical characteristic are reported byGil et al., “Stimuli-responsive polymers and their bioconjugates,” Prog.Polym. Sci., 29, 1173-1222 (2004) which is incorporated by referenceherein. A thermally responsive polymer is one example of thesematerials. A thermally responsive material is a material in which aphysical property is altered in response to a change in temperature inthe surrounding environment or system. A thermally responsive polymermay change from a hydrophilic state to hydrophobic state when thetemperature of the system or its surroundings rises above a lowercritical solution temperature (LCST). When in a hydrophilic state, thepolymer chains become swollen. Conversely, in a hydrophobic state, thepolymer chains collapse, and the polymer becomes insoluble in water. Inmost cases, the process can be reversible.

One embodiment of the present invention is directed to a thermallyresponsive nanofiber. The thermally responsive nanofiber can be used tomodify a surface of a substrate to provide a functionalized surface.More particularly, the thermally responsive nanofiber can be used toprovide a thermally responsive surface on a substrate. The physicalproperty of the thermally responsive nanofiber modified surface of thesubstrate changes in response to a change in temperature in the system.Biologically active materials can be immobilized on the nanofibermodified surface by reacting with the functional groups accessible orexposed on the surface of the substrate. Typically, the biologicallyactive materials retain all or a portion of their bioactivity afterhaving been immobilized on the thermally responsive nanofiber modifiedsurface. The ability of biologically active materials to bond with thesurface of the substrate can be affected depending on the physical stateof the modified surface. Thus, by controlling the temperature of themodified surface, the ability to bind to a biological material can becontrolled.

According to one embodiment of the present invention the thermallyresponsive nanofiber includes a thermally responsive polymer, abiologically active material, and a cross-linking agent having at leasttwo latent reactive activatable groups. The thermally responsivenanofiber can be used to modify the surface of a substrate by bondingthe nanofiber to the surface by the formation of a covalent bond betweenthe surface of the substrate and the nanofiber. At least one of thelatent reactive activatable groups undergoes activation when subjectedto a suitable energy source to form a covalent bond between the surfaceof the substrate and the thermally responsive nanofiber. The remaininglatent reactive group(s) are left accessible or exposed on the surfaceof the substrate. The biologically active material included, in thenanofiber or the accessible or exposed latent reactive groups on thesurface may be used for further surface modification of the substrate.

A number of processing techniques such as drawing, template synthesis,phase separation, self-assembly or electrospinning have been used toprepare nanofibers.

For example, a thermally responsive nanofiber can be formed byelectrospinning a fiber-forming combination that includes a thermallyresponsive polymer, a biologically active material, and a cross-linkingagent having at least two latent reactive activatable groups.Electrospinning, generally involves the introduction of a polymer orother fiber-forming solution or liquid into an electric field, so thatthe solution or liquid is caused to produce fibers. When a strongelectrostatic field is applied to a fiber-forming combination held in asyringe with a capillary outlet, a pendant droplet of the fiber-formingmixture from the capillary outlet is deformed into a Taylor cone. Whenthe voltage surpasses a threshold value, the electric forces overcomethe surface tension, on the droplet, and a charged jet of the solutionor liquid is ejected from the tip of the Taylor cone. The ejected jetthen moves toward a collecting metal screen that acts as acounterelectrode having a lower electrical potential. The jet is splitinto small charged fibers or fibrils and any solvent present evaporatesleaving behind a nonwoven fabric mat formed on the screen.

In one embodiment, electrostatically spun fibers can be produced havingvery thin diameters. Parameters that influence the diameter,consistency, and uniformity of the electrospun fibers include thethermally responsive polymer, the molecular weight of the polymer; thecross-linker concentration (loading) in the fiber-forming mixture, theflow rate of the polymer solution, the applied voltage, and the needlecollector distance. According to one embodiment of the presentinvention, a stimuli responsive nanofiber has a diameter ranging fromabout 1 nm to about 100 μm. In other embodiments, the stimuli responsivenanofiber has a diameter in a range of about 1 nm to about 1000 nm.Further, the nanofiber may have an aspect ratio in a range of about atleast 10 to about at least 100. It will be appreciated that, because ofthe very small diameter of the fibers, the fibers have a high surfacearea per unit of mass. This high surface area to mass ratio permitsfiber-forming material solutions to be transformed from solvatedfiber-forming materials to solid nanofibers in fractions of it second.

The stimuli responsive polymer used to form the nanofiber may beselected from any stimuli responsive, fiber-forming material that iscompatible with the cross-linking agent. In one embodiment, a selectedthermally responsive polymer should be capable of undergoing a rapidchange from a first physical property to a second physical property whenthe temperature of the system has risen above a lower critical solutiontemperature. Exemplary thermally responsive, fiber forming polymersinclude, but are not limited to, poly(isopropylacrylamide) and mixturesand copolymers thereof. Other thermally responsive polymers includerandom copolymers of 2-(2-methoxyethoxy)ethyl methacrylate andoligo(ethylene glycol) methacrylate.

According to one embodiment of the present invention, the thermallyresponsive polymer is poly(isopropylacrylamide).Poly(isopropylacrylamide) changes from a primarily hydrophobic state toa primarily hydrophilic state upon reaching a lower critical solutiontemperature of approximately 20 to 32° C. Poly-N-isopropylacrylamide(PIPAAm) has been one of the most studied thermo-responsive polymer notonly because it displays a low critical solution temperature (LCST) ofaround 32° C., close to body temperature, but also because its LCST isrelatively insensitive to environmental conditions. Slight variations ofpH, concentration or chemical environment affect the LCST by only a fewdegrees. The main mechanism of PIPAAm's aqueous phase separation is thethermally induced release of water molecules bound to polymer isopropylside groups, resulting in intra- and intermolecular hydrophobicinteractions between isopropyl groups above the LCST.

The inclusion of cross-linking agents within the composition forming thethermally responsive nanofiber, allows the thermally responsivenanofiber to be compatible with a wide range of support surfaces. Thelatent reactive cross-linking agents can be used alone or in combinationwith other materials to provide a desired surface characteristic.

Suitable cross-linking agents include either monomeric (small moleculemateials) or polymeric materials having at least two latent reactiveactivatable groups that are capable of forming covalent bonds with othermaterials when subjected to a source of energy such as radiation,electrical or thermal energy. In general, latent reactive activatablegroups are chemical entities that respond to specific applied, externalenergy or stimuli to generate active species with resultant covalentbonding to an adjacent chemical structure. Latent reactive groups arethose groups that retain their covalent bonds under storage conditionsbut that form covalent bonds with other molecules upon activation by anexternal energy source. In some embodiments, latent reactive groups formactive species such as free radicals. These free radicals may includenitrenes, carbine or excited states of ketones upon absorption ofexternally applied electric, electromagnetic or thermal energy. Variousexamples of known latent reactive groups are reported U.S. Pat. No.4,973, U.S. Pat. No. 5,258,041 and U.S. Pat. No. 5,563,056.

Eight commercially available multifunctional photocrosslinkers based ontrichloromethyl triazine are available either from Aldrich Chemicals,Produits Chimiques Auxilíaìres et de Syntheses, (Longjumeau, France),Shin-Nakamara Chemical, Midori Chemicals Co., Ltd. or Panchim S.A.(France). The eight compounds include 2,4,6-tris(trichloromethyl)-1,3,5triazine, 2-(methyl)-4,6-bis(trichloromethyl)-1,3,5-triazin(4-methoxynaphthyl)-4,6-bis(trichloromethyl)-1,3,5-triazine,2-(4-ethoxynaphthyl)-4,6 bis(trichloromethyl)-1,3,5-triazine,4-(4-carboxylphenyl)-2,6-bis(trichloromethyl)-1,3,5-triazine,2-(4-methoxyphenyl)-4,6-bis(trichloromethyl)-1,3,5-triazine,2-(1-ethen-2,2′-furyl)-4,6-bis(trichloromethyl)-1,3,5-triazine and2-(4-methoxystyryl)-4,6-bis(trichloromethyl)-1,3,5 triazine.

In some embodiments, the latent reactive groups are the same, while inother embodiments the latent reactive groups may be different. Forexample, the latent reactive groups may be two different groups that areboth activated by radiation. In other embodiments one latent reactivegroup may by activated by radiation while another latent reactive groupmay be activated by heat. Suitable cross-linking agents include bi-,tri-and multi-functional monomeric and polymeric materials.

Latent reactive groups that are reactive to thermal or heat energyinclude a variety of reactive moieties and may include known compoundsthat decompose thermally to than reactive species that will then formcovalent bonds. The covalent bonds allow the cross-linking to bind toadjacent materials. Suitable thermally-reactive groups typically have apair of atoms having a heat sensitive or labile bond. Heat labile bondsinclude oxygen-oxygen bonds such as peroxide bonds, nitrogen-oxygenbonds, and nitrogen-nitrogen bonds. Such bonds will react or decomposeat temperatures in a range of not more than 80-200° C.

Both thermally generated carbenes and nitrenes undergo a variety ofchemical reactions, including carbon bond insertion, migration, hydrogenabstraction, and dimerization. Examples of carbene generators includediazirines and diazo-compounds. Examples of nitrene generators includearyl azides, particularly perfluorinated aryl azides, acyl azides, andtriazolium ylides. In addition, groups that upon heating form reactivetriplet states, such as dioxetanes, or radical anions and radicalcations may also be used to form the thermally-reactive group.

In one embodiment the thermally-reactive group of the cross-linkingagent includes a peroxide —(O—O)— group. Thermally-reactiveperoxide-containing groups include, for example, thermally-reactivediacyl peroxide groups, thermally-reactive peroxydicarbonate groups,thermally-reactive dialkylperoxide groups, thermally-reactiveperoxyester groups, thermally-reactive peroxyketal groups, andthermally-reactive dioxetane groups.

Dioxetanes are four-membered cyclic peroxides that react or decompose atlower temperatures compared to standard peroxides due to the ring strainof the molecules. The initial step in the decomposition of dioxetanes iscleavage of the O—O bond, the second step breaks the C—C bond creatingone carbonyl in the excited triplet state, and one in an excited singletstate. The excited triplet state carbonyl can extract a hydrogen from anadjacent material, forming two radical species, one on the adjacentmaterial and one on the carbon of the carbonyl with the oxygen and willform a new covalent bond between the thermally reactive dioxetane andthe adjacent material.

Representative thermally reactive moieties are reported in US20060030669 and other representative thermal latent reactive groups arereported in U.S. Pat. No. 5,258,041. Both of these documents are herebyincorporated by reference.

Latent reactive groups that are reactive to electromagnetic radiation,such as ultraviolet or visible radiation, are typically referred to asphotochemical reactive groups.

The use of latent reactive activatable species in the form of latentreactive activatable aryl ketones is useful. Exemplary latent reactiveactivatable aryl ketones include acetophenone, benzophenone,anthraquinone, anthrone, anthrone-like heterocycles (i.e., heterocyclicanalogs of anthrone such as those having N, O, or S in the 10-position),and their substituted (e.g., ring substituted) derivatives. Examples ofaryl ketones include heterocyclic derivatives of anthrone, includingacridone, xanthone, and thioxanthone, and their ring substitutedderivatives. In particular, thioxanthone, and its derivatives, havingexcitation energies greater than about 360 nm are useful.

The functional groups of such ketones are suitable since they arereadily capable of undergoing an activation/inactivation/reactivationcycle. Benzophenone is an exemplary photochemically reactive activatablegroup, since it is capable of photochemical excitation with the initialformation of an excited singlet state that undergoes intersystemcrossing to the triplet state. The excited triplet state can insert intocarbon-hydrogen bonds by abstraction of a hydrogen atom (from a supportsurface, for example), thus creating a radical pair. Subsequent collapseof the radical pair leads to formation of a new carbon-carbon bond. If areactive bond carbon-hydrogen) is not available for bonding, theultraviolet light-induced excitation of the benzophenone group isreversible and the molecule returns to ground state energy level uponremoval of the energy source. Photochemically reactive activatable arylketones such as benzophenone and acetophenone are of particularimportance inasmuch as these groups are subject to multiple reactivationin water and hence provide increased coating efficiency.

In some embodiments of the invention, photochemically reactivecross-linking agents may be derived from three different types ofmolecular families. Some families include one or more hydrophilicportions, i.e., a hydroxyl group (that may be protected), amines, alkoxygroups, etc. Other families may include hydrophobic and amphiphilicportions. In one embodiment, the family has the formula:

L-((D-T-C(R¹)(XP)CHR²GR³C(═O)⁴))_(m).

L is a linking group. D is O, S, SO, SO₂, NR⁵ or CR⁶R⁷. T is(—CH₂—)_(x), (—CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂—O—)_(x) or(—CH₂CH₂CH₂CH₂—O—)_(x). R¹ is a hydrogen atom, an alkyl, alkyloxyakylaryl, aryloxyalkyl or aryloxyaryl group. X is O, S, or NR⁸R⁹. P is ahydrogen atom or a protecting group, with the proviso that P is absentwhen X is NR⁸R⁹. R² is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl,aryloxylalkyl or aryloxyaryl group. G is O, S, SO, SO₂, NR¹⁰,(CH₂)_(t)—O— or C═O. R³ and R⁴ are each independently an alkyl, aryl,arylalkyl, heteroaryl, or a heteroarylalkyl group or when R³ and R⁴ aretethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s),(—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂ 13 )_(s),(—CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)(—CH₂—)_(s). R⁵ and R¹⁰ areeach independently a hydrogen atom or an alkyl, aryl, or arylalkylgroup. R⁶ and R⁷ are each independently a hydrogen atom, an alkyl, aryl,arylalkyl, heteroaryl or heteroarylalkyl group. R⁸ and R⁹ are eachindependently a hydrogen atom, an alkyl, aryl, or arylalkyl group, R isa hydrogen atom, an alkyl group or an aryl group, q is an integer from 1to about 7, r is an integer from 0 to about 3, s is an integer from 0 toabout 3, m is an integer from 2 to about 10, t is an integer from 1 toabout 10 and x is an integer from 1 to about 500.

In one embodiment, L is a branched or unbranched alkyl chain havingbetween about 2 and about 10 carbon atoms.

In another embodiment, D is an oxygen atom (O).

In still another embodiment, T is (—CH₂—)_(x) or (—CH₂CH₂—O—), and x is1 or 2.

In still yet another embodiment, R¹ is a hydrogen atom.

In yet another embodiment, X is an oxygen atom, O, and P is a hydrogenatom.

In another embodiment, R² is a hydrogen atom.

In still another embodiment, G is an oxygen atom, O.

In still yet another embodiment, R³ and R⁴ are each individually arylgroups, which can be further substituted, and m is 3.

In one particular embodiment, L is

D is O, T is (—CH₂—)_(x,R) ¹ is a hydrogen atom, X is O, P is a hydrogenatom, R² is a hydrogen atom, G is O, R³ and R⁴ are phenyl groups, m is 3and x is 1.

In yet another particular embodiment, L is (—CH₂—)_(y), D is O, T is(—CH₂—)_(x), R¹ is a hydrogen atom, X is O, P is a hydrogen atom, R² isa hydrogen atom, G is O, R³ and R⁴ are phenyl groups, m is 2, x is 1 andy is an integer from 2 to about 6, and in particular, y is 2, 4 or 6.

In certain embodiments, x is an integer from about 1 to about 500, moreparticularly from about 1 to about 400, from about 1 to about 250, fromabout 1 to about 200, from about 1 to about 150, from about 1 to about100, from about 1 to about 50, from about 1 to about 25 or from about 1to about 10.

In another embodiment, the family has the formula:

L-((T-C(R¹)(XP)CHR²GR³C(═O)R⁴))_(m).

and L, T, R¹, X, P, R², G, R³, R⁴, R⁸, R⁹, R¹⁰, q, r, s, in, t and x areas defined above.

In one embodiment, I, has a formula according to structure (1):

A and J are each independently a hydrogen atom, an alkyl group, an arylgroup, or together with B form a cyclic ring, provided when A and J areeach independently a hydrogen atom, an alkyl group, or an aryl groupthen B is not present, B is NR¹¹, O, or (—CH₂—)₂, provided when A, B andJ form a ring, then A and J are (—CH₂—)₂ or C═O, R¹¹ is a hydrogen atom,an alkyl group, an aryl group or denotes a bond with T, each zindependently is an integer from 0 to 3 and provided when either A or Jis C═O, then B is NR¹¹, O, or (—CH₂—)_(z) and z must be at least 1

In another embodiment, T is —CH₂—.

In another embodiment, the family has the formula:L-((GTZR³C(═O)R⁴))_(m), and L. T, G, R³, R⁴, R¹⁰, R, q, r, s, m, t and xare as defined above. Z can be a C═O, COO or CONH when T is (—CH₂—)_(x).

In one embodiment, L has a formula according to structure (I):

and A, B, J, R¹¹, and z are as defined above.

In another embodiment, L has a formula according to structure (II):

R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ are each independently a hydrogen atom, analkyl or aryl group or denotes a bond with T, provided at least two ofR¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ are bonded with T and each K, independentlyis CH or N.

In another embodiment, the family has the formula:

L-((TGQR³C(═O)R⁴))_(m).

L, G, R³, R⁴, R¹⁰, R, q, r, s, m, t and x are as defined above. T is(—CH₂—)_(x), (—CH₂CH₂—O—x, (—CH₂CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂CH₂—O—)_(x)or forms a bond. Q is (—CH₂—)_(p), (—CH₂CH₂—O—)_(p), (—CH₂CH₂CH₂O—)_(p)or (—CH₂CH₂CH₂CH₂—O—)_(p) and p is an integer from 1 to about 10.

In one embodiment, L has a formula according to structure (I):

A, B, J, R¹¹, and z are as defined above.

In another embodiment, L has a formula according to structure (II):

R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ are each independently a hydrogen atom, analkyl or aryl goup or denotes a bond with T, provided at least two ofR¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ are bonded with T and each K, independentlyis CH or N.

In still yet another embodiment, compounds of the present inventionprovide that R³ and R⁴ are both phenyl groups and are tethered togethervia a CO, a S or a CH₂.

In yet another embodiment, compounds of the present invention providewhen R³ and R⁴ are phenyl groups, the phenyl groups can eachindependently be substituted with at least one alkyloxyalkyl group, suchas CH₃O—(CH₂CH₂O—)_(n)—, or CH₃O(—CH₂CH₂CH₂O—) n-a hydroxylated alkoxygroup, such as HO—CH₂CH₂O—, HO(—CH₂CH₂O—)_(n)— or HO(—CH₂CH₂CH₂O—)_(n)—,etc. wherein n is an integer from 1 to about 10.

In another embodiment the family has the formula:

L-(((—CH₂—)_(xx)C(R¹)((G)R³C(═O)R⁴)₂)_(m).

L, each R, R¹, each G, each R³, each R⁴, each R¹⁰, each q, each r, eachs, each t and m are as defined above and xx is an integer from 1 toabout 10.

In one embodiment, L has a formula according to structure (I):

A, B, J, R¹¹, and z are as defined above.

In another embodiment, A and B are both hydrogen atoms.

In still another embodiment, xx is 1.

In yet another embodiment, R¹ is

In still yet another embodiment, G is (—CH₂—)_(t)O— and t is 1.

In another embodiment, R³ and R⁴ are each individually aryl groups.

In still yet another embodiment, xx is 1, R¹ is H, each G is(—CH₂—)_(t)O—, t is 1 and each of R³ and R⁴ are each individually arylgroups.

In another embodiment of the invention, the family has the formula:

L-((—C(R¹)(XP)CHR²GR³C(═O)R⁴)_(m).

L, R, R¹, R², R³, R⁴, R⁸, R⁹, R¹⁰, X, P, G, q, r, s, t, and m are asdefined above.

In one embodiment, L is

and R²⁰ and R²¹ are each individually a hydrogen atom, an alkyl group oran aryl group.

In another embodiment, R¹ is H.

In still another embodiment, wherein X is O.

In yet another embodiment, P is H.

In still yet another embodiment, R² is H.

In another embodiment, G is (—CH₂—)_(t)O— and t is 1.

In still another embodiment, R³ and R⁴ are each individually arylgroups.

In yet another embodiment, R¹ is H, X is O, P is H, R² is H, G is(—CH₂—)_(t)O—, t is 1, R³ and R⁴ are each individually aryl groups andR²⁰ and R²¹ are both methyl groups.

In yet another embodiment, the present invention provides a family ofcompounds having the formula:

L-((GR³C(═O)R⁴))_(m).

L is a linking group; G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; R³and R⁴ are each independently an alkyl, aryl, arylalkyl, heteroaryl, oran heteroarylalkyl group or when R³ and R⁴ are tethered together via(—CH₂—)_(q), (—CH₂—)_(t)C═O(—CH₂—)_(s), (—CH₂—)_(t)S(—CH₂—)_(s)(—CH₂—)_(r)S═O(—CH₂—)_(s) or (—CH₂—)_(t)S(O)₂(—CH₂—)_(s),(—CH₂—)_(r)NR(—CH₂—)_(s); R¹⁰ is a hydrogen atom or an alkyl, aryl, oran arylalkyl group; R is a hydrogen atom, an alkyl or an aryl group; qis an integer from 1 to about 7; r is an integer from 0 to about 3; s isan integer from 0 to about 3; in is an integer from 2 to about 10; and tis an integer from 1 to about 10.

In one embodiment, L is

In another embodiment, G is C═O.

In still another embodiment, R³ and R⁴ are each individually arylgroups.

In yet another embodiment, G is C═O and R³ and R⁴ are each individuallyaryl groups.

“Alkyl” by itself or as part of another substituent refers to asaturated or unsaturated branched, straight-chain or cyclic monovalenthydrocarbon radical having the stated number of carbon atoms (i.e.,C₁-C₆ means one to six carbon atoms) that is derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane, alkeneor alkyne, Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, 1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, 1,3-dien-2-yl, cyclobut-1-en-1-yl,cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Where specific levelsof saturation are intended, the nomenclature “alkanyl,” “alkenyl” and/or“alkynyl” is used, as defined below. “Lower alkyl” refers to alkylgroups having from 1 to 6 carbon atoms.

“Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane. Typical alkanyl groups include, but are not limited to,methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl,butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like.

“Alkenyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon double bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkene. The group may be in eitherthe cis or trans conformation about the double bond(s). Typical alkenylgroups include, but are not limited to, ethenyl; propenyls such asprop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,cycloprop-1-en-1-yl; -cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, and thelike.

“Alkyloxyalkyl” refers to a moiety having two alkyl groups tetheredtogether via an oxygen bond. Suitable alkyloxyalkyl groups includepolyoxyalkylenes, such as polyethyleneoxides, polypropyleneoxides, etc.that are terminated with an alkyl group, such as a methyl group. Ageneral formula for such compounds can be depicted as R³- (OR″)_(n) or(R′O)_(n)—R″ wherein a is an integer from 1 to about 10, and R′ and R″are alkyl or alkylene groups.

“Alkynyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon triple bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkyne. Typical alkynyl groupsinclude, but are not limited to, ethynyl; propynyls such asprop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

“Alkyldiyl” by itself or as part of another substituent refers to asaturated or unsaturated, branched, straight-chain or cyclic divalenthydrocarbon group having the stated number of carbon atoms (i.e., C₁-C₆means from one to six carbon atoms) derived by the removal of onehydrogen atom from each of two different carbon atoms of a parentalkane, alkene or alkyne, or by the removal of two hydrogen atoms from asingle carbon atom of a parent alkane, alkene or alkyne. The twomonovalent radical centers or each valency of the divalent radicalcenter can form bonds with the same or different atoms. Typicalalkyldiyl groups include, but are not limited to, methandiyl; ethyldiylssuch as ethan-1,1-diyl, ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl;propyldiyls such as propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl,propan-1,3-diyl, cyclopropan-1,1-diyl, -cyclopropan-1,2-diyl,prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl,prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as,butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl,butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien-1,4-diyl,cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; andthe like. Where specific levels of saturation are intended, thenomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Whereit is specifically intended that the two valencies be on the same carbonatom, the nomenclature “alkylidene” is used. A “lower alkyldiyl” is analkyldiyl group having from 1 to 6 carbon atoms. In some embodiments thealkyldiyl groups are saturated acyclic alkanyldiyl groups in which theradical centers are at the terminal carbons, e.g., methandiyl (methano);ethan-1,2-diyl (ethano); propan-1,3-diyl (propano); butan-1,4-diyl(butano); and the like (also referred to as alkylenes, defined infra).

“Alkylene” by itself or as part of another substituent refers to astraight-chain saturated or unsaturated alkyldiyl group having twoterminal monovalent radical centers derived by the removal of onehydrogen atom from each of the two terminal carbon atoms ofstraight-chain parent alkane, alkene or alkyne. The location of a doublebond or triple bond, if present, in a particular alkylene is indicatedin square brackets. Typical alkylene groups include, but are not limitedto, methylene (methano); ethylenes such as ethano, etheno, ethyno;propylenes such as propano, prop[1]eno, propa[1,2]dieno, prop[1]yno,etc.; butylenes such as butano, but[1]eno, but[2]eno, buta[1,3]dieno,but[1]yno, but[2]yno, buta[1,3]diyno, etc.; and the like. Where specificlevels of saturation are intended, the nomenclature alkano, alkenoand/or alkyno is used. In some embodiments, the alkylene group is(C₁-C₆) or (C₁-C₃) alkylene. Other embodiments include straight-chainsaturated alkano groups, e.g., methano, ethano, propano, butano, and thelike.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon group having the stated number of carbonatoms (i.e., C₅-C₁₅ means from 5 to 15 carbon atoms) derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limit to,groups derived from aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, coronene, fluoranthene,fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene,indane, indene, naphthalene, octacene, octaphene, octalene, ovalene,penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene,phenanthrene, picene, pleiadene pyrene, pyranthrene, rubicene,triphenylene, trinaphthalene, and the like, as well as the various hydroisomers thereof. In some embodiments, the aryl group is (C₅-C₁₅) arylor, alternatively, (C₅-C₁₀) aryl. Other embodiments include phenyl andnaphthyl.

“Arylalkyl” by itself or as part of another substituent refers to anacyclic alky radical in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylalkenyl and/orarylalkynyl is used. In some embodiments, the arylalkyl group is(C₇-C₃₀) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C₁-C₁₀) and the aryl moiety is (C₆-C₂₀) or,alternatively, an arylalkyl group is (C₇-C₂₀) arylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C₁-C₈) andthe aryl moiety is (C₆-C₁₂).

“Aryloxyalkyl” refers to a moiety having an aryl group and an alkylgroup tethered together via an oxygen bond. Suitable aryloxy alkylgroups include phenyloxyalkylenes, such as methoxyphenyl orethoxyphenyl.

“Cycloalkyl” by itself or as part of another substituent refers to acyclic version of an “alkyl” group. Typical cycloalkyl groups include,but are not limited to, cyclopropyl; cyclobutyls such as cyclobutanyland cyclobutenyl; cyclopentyls such as cyclopentanyl and cycloalkenyl;cyclohexyls such as cyclohexanyl and cyclohexenyl; and the like.

“Cycloheteroalkyl” by itself or as part of another substituent refers toa saturated or unsaturated cyclic alkyl radical in which one or morecarbon atoms (and any associated hydrogen atoms) are independentlyreplaced with the same or different heteroatom. Typical heteroatoms toreplace the carbon atom(s) include, but are not limited to, N, P, O, Sor Si. Where a specific level of saturation is intended, thenomenclature “cycloheteroalkanyl” or “cycloheteroalkenyl” is used.Typical cycloheteroalkyl groups include, but are not limited to, groupsderived from epoxides, imidazolidine, morpholine, piperazine,piperidine, pyrazolidine, pyrrolidine, quinuclidine, and the like.

“Halogen” or “Halo” by themselves or as part of another substituent,unless otherwise stated, refer to fluoro, chloro, bromo and iodo.

“Haloalkyl” by itself or as part of another substituent refers to analkyl group in which one or more of the hydrogen atoms are replaced witha halogen. Thus, the term “haloalkyl” is meant to includemonohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls.For example, the expression “(C₁-C₂) haloalkyl” includes fluoromethyl,difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl,1,2-difluoroethyl, 1,1,1-trifluoroethyl or perfluoroethyl.

“Heteroalkyl, Heteroalkanyl, Heteroalkenyl, Heteroalkynyl” by itself oras part of another substituent refer to alkyl, alkanyl, alkenyl andalkynyl radical, respectively, in which one or more of the carbon atoms(and any associated hydrogen atoms) are each independently replaced withthe same or different heteroatomic groups. Typical heteroatomic groupsinclude, but are not limited to, —O—, —S—, —O—O—, —S—S—, —O—S—, —NR′═,N—N═, —N═N—, —N═N—NR′, —PH—, —P(O)₂—, —O—P(O)₂—, —S(O)—, —S(O)₂—, —SnH₂—and the like, where R′ is hydrogen, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, aryl or substituted aryl.

“Heteroaryl” by itself or as part of another substituent, refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a parent heteroaromatic ring system. Typicalheteroaryl groups include, but are not limited to, groups derived fromacridine, arsindole, carbazole, β-carboline, benzoxazine, benzimidazole,chromane, chromene, cinnoline, furan, imidazole, indazole, indole,indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like. The heteroaryl group may befrom 5-20 membered heteroaryl or, alternatively, from 5-10 memberedheteroaryl. In some embodiments, the heteroaryl groups are those derivedfrom thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine,quinoline, imidazole, oxazole and pyrazine.

“Heteroarylalkyl” by itself or as part of another substituent refers toan acyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced with aheteroaryl group. Where specific alkyl moieties are intended, thenomenclature heteroarylalkanyl, heteroarylakenyl and/orheteroarylalkynyl is used. In some embodiments, the heteroarylalkylgroup is a 6-21 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is (C₁-C₆) alkyl and theheteroaryl moiety is a 5-15-membered heteroaryl. In other embodiments,the heteroarylalkyl is a 6-13 membered heteroarylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety is (C₁-C₃) alkyl and the heteroarylmoiety is a 5-10 membered heteroaryl.

“Hydroxyalkyl” by itself or as part of another substituent refers to analkyl group in which one or more of the hydrogen atoms are replaced witha hydroxyl substituent. Thus, the term “hydroxyalkyl” is meant toinclude monohydroxyalkyls, dihydroxyalkyls or trihydroxyalkyls.

“Parent Aromatic Ring System” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated π electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, fluorene, indane, indene, phenalene,tetrahydronaphthalene, etc. Typical parent aromatic ring systemsinclude, but are not limited to, aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene hexalene, indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, tetrahydronaphthalene, triphenylene, trinaphthalene, and thelike, as well as the various hydro isomers thereof.

“Parent Heteroaromatic Ring System” refers to a parent aromatic ringsystem in which one or more carbon atoms (and any associated hydrogenatoms) are independently replaced with the same or different heteroatom.Typical heteroatoms to replace the carbon atoms include, but are notlimited to, N, P, O, S, Si, etc. Specifically included within thedefinition of “parent heteroaromatic ring systems” are fused ringsystems in which one or more of the rings are aromatic and one or moreof the rings are saturated or unsaturated, such as, for example,arsindole, benzodioxan, benzofuran, chromane, chromene, indole,indoline, xanthene, etc. Typical parent heteroaromatic ring systemsinclude, but are not limited to, arsindole, carbazole, β-carboline,chromane, chromene, cinnoline, furan, imidazole, indazole, indole,indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like.

“Leaving group” is a group that is displaced during a reaction by anucleophilic reagent. Suitable leaving groups include S(O)₂Me, —SMe orhalo (e.g., F, Cl, Br, I).

“Linking group” is a group that serves as an intermediate locus betweentwo or more end groups. The nature of the linking group can vary widely,and can include virtually any combination of atoms or groups useful forspacing one molecular moiety from another. For example, the linker maybe an acyclic hydrocarbon bridge (e.g., a saturated or unsaturatedalkyleno such as methano, ethano, etheno, propano, prop[1]eno, butano,but[1]eno, but[2]eno, buta[1,3]dieno, and the like), a monocyclic orpolycyclic hydrocarbon bridge (e.g., [1,2]benzeno, [2,3]naphthaleno, andthe like), a simple acyclic heteroatomic or heteroalkyldiyl bridge(e.g., —O—, —S—, —S—O—, —NH—, —PH—, —C(O)—, —C(O)NH—, —S(O)—, —S(O)₂—,—S(O)NH—, —S(O)₂NH—, —O—CH₂—, —CH₂—, —CH₂—O—CH₂—, —O—CH═CH—CH₂—, and thelike), a monocyclic or polycyclic heteroaryl bridge (e.g., [3,4]furano,pyridino, thiopheno, piperidino, piperazino, pyrazidino pyrrolidino, andthe like) or combinations of such bridges.

“Protecting group” is a group that is appended to, for example, ahydroxyl oxygen in place of a labile hydrogen atom. Suitable hydroxylprotecting group(s) include esters (acetate, ethylacetate), ethers(methyl, ethyl), ethoxylated derivatives (ethylene glycol, propyleneglycol) and the like that can be removed under either acidic or basicconditions so that the protecting group is removed and replaced with ahydrogen atom. Guidance for selecting appropriate protecting groups, aswell as synthetic strategies for their attachment and removal, may befound, for example, in Greene & Wuts, Protective Groups in OrganicSynthesis, 3d Edition, John Wiley & Sons, Inc., New York (1999) and thereferences cited therein (hereinafter “Greene & Wuts”).

Plastics or porous membranes such as polyolefins, polystyrenes,poly(methyl)methacrylates, polyacrylonitriles, poly(vinylacetates), poly(vinyl alcohols), chlorine-containing polymers such as poly(vinyl)chloride, polyoxymethylenes, polycarbonates, polyamides, polyimides,polyurethanes, phenolics, amino-epoxy resins, polyesters, silicones,cellulose-based plastics, fluoropolymers and rubber-like plastic can allbe used as supports, providing surfaces that can be modified asdescribed herein. In addition, supports such as those formed ofpyrolytic carbon, parylene coated surfaces, and silylated surfaces ofglass, ceramic, or metal are suitable for surface modification.

The method of the present invention may involve the attachment of abiologically active material to a support surface. For example, athermally responsive nanofiber including a cross-linking agent isprovided having two or more latent reactive activable groups in thepresence of a support surface. According to an alternative embodiment,the nanofiber may also include a biologically active material or afunctional polymer that is reactive with a biologically active material.At least one of the latent reactive groups is activated and covalentlybonded to the surface. The remaining latent reactive groups are allowedto remain in their inactive state and are later activated in order tobind a biologically active material or a functional polymer in order toattach the biologically active material to the surface of the substrate.

A functional polymer is a polymer having one or more functional groupsthat will react with a biologically active material. Representativefunctional groups include carboxy, ester, epoxy, hydroxyl, amido, amino,thio N-hydroxy succinimide, isocyante, anhydride, azide, aldehyde,cyanuryl chloride or phosphine groups that will react with abiologically active material.

Alternatively, the biologically active material or functional polymerprovided in the thermally responsive nanofiber composition may bind to asecond biological material in order to attach the second biologicalmaterial to the surface of the substrate through manipulation of thephysical properties of the support surface via, for example, theapplication or removal of heat from the system.

The steps of the method can be performed in any suitable order. Forexample, a thermally responsive nanofiber including a thermallyresponsive polymer and cross-linking agent, as described herein, may bephysically absorbed in or adsorbed to a suitable support surface byhydrophobic interactions. Upon photoactivation at least one of thephotoactivatable groups (e.g., benzophenone groups) undergoes covalentbond formation at the support surface. With the absence of abstractablehydrogens in the proximity of the remaining unbonded photoactivatablegroup(s), and removal of the photoactivation source, thephotoactivatable group returns from an excited state to a ground state.These remaining photoactivatable groups are then capable of beingreactivated when a biologically active material intended forimmobilization is present, and when the treated surface is exposed toanother round of illumination. This method can be described as a“two-step” approach, where the thermally responsive nanofiber is appliedin the first step to create a latent reactive surface, and in the secondstep, the biologically active material is added for attachment to theactivated surface.

Alternatively, the method, described as a “one-step” method, providesthat the thermally responsive nanofibers of the present invention aremixed together with the biologically active material to form acomposition. The resultant composition is used to surface modifymaterials in a single photoactivation step. In this case,photoactivation triggers not only covalent bond formation of at leastone photoactivatable group with the surface of the substrate, but alsosimultaneously triggers covalent bond formation with any adjacentbiologically active materials residing on the surface.

In an alternative embodiment, the thermally responsive nanofiber isformed from a combination or mixture including a thermally responsivepolymer, a cross-linking agent having at least two latent activatablegroups, and a biologically active material. At least one of the latentreactive groups undergoes covalent bond formation at the support surfaceto bond the nanofiber to the surface of the substrate. The remaininglatent reactive group(s) can undergo photoactivation to react with asecond biologically active material. Alternatively, the biologicallyactive material incorporated into the nanofiber can itself react with asecond biologically active material to provide for furtherfunctionalization of the substrate.

In another alternative method, the thermally responsive nanofibers ofthe present invention are used to pretreat a substrate surface prior tothe application and bonding of molecules that have themselves beenfunctionalized with latent reactive groups. This method is useful insituations where a particularly difficult substrate requires maximalcoating durability. In this manner, the number of covalent bonds formedbetween the substrate surface and the target molecule derivatized withlatent reactive groups can typically be increased, as compared tosurface modification with a desired latent reactive group-containingtarget molecule alone.

After the surface of a substrate has been coated or treated with thethermally responsive nanofibers of the present invention, the thermallyresponsive surface can then be fine toned by the application or removalof heat to the system to selectively bind and release a biologicalmaterial of interest. Heat can be applied to the system to transitionthe thermally responsive nanofiber bound to the surface from ahydrophilic state to a hydrophobic state. In a hydrophobic state at atemperature higher than LCST, the polymer chains collapse and thesurface becomes hydrophobic. In this state the thermally responsivenanofiber surface may attract or repel select target molecules.Alternativly, heat can also be removed from the system by cooling thesubstrate below the LCST. Once cooled, the thermally responsivenanofiber may revert back to its initial hydrophilic state, once againshowing an altered affinity for a particular target molecule.

Suitable biologically active or target molecules for use in the presentinvention encompass a diverse group of materials or substances. Thesematerials may be used in either an underivatized form or previouslyderivatized. Moreover, target molecules can be immobilized singly or incombination with other types of target molecules.

Target molecules can be immobilized to the surface after (e.g.,sequentially) the surface has been primed with the thermally responsivenanofibers of the present invention. Alternatively, target molecules areimmobilized during (e.g., simultaneously with) attachment of thethermally responsive nanofibers to the surface of the substrate.

Typically, target molecules are selected so as to confer particulardesired properties to the surface and/or to the device or articlebearing the surface. According to one embodiment of the presentinvention, the target molecule or material is a biologically activematerial. Biologically active materials which may be immobilized on thesurface of the nanofiber modified substrate, or alternatively, providedas a part of the nanofiber composition, generally include, but are notlimited to, the following: enzymes, proteins, carbohydrates, nucleicacids, and mixtures thereof. Further examples of suitable targetmolecules, including biological materials, and the surface propertiesthey are typically used to provide, is represented by the followingnonlimiting list.

TARGET MOLECULE FUNCTIONAL ACTIVITY Synthetic Polymers Sulfonicacid-substituted Lubricity, negatively charged surface, Polyacrylamidehydrophilicity Polyacrylamide Lubricity, protein repulsion,hydrophilicity Polyethylene glycol Lubricity, cell and proteinrepulsion, hydrophilicity Polyethyleneimine Positively charged surfacePolylactic acid Bioerodible surface Polyvinyl alcohol Lubricity,hydrophilicity Polyvinyl pyrrolidone Lubricity, hydrophilicityQuaternary Lubricity positively charged surface amine-substitutedpolyacrylamide Silicone Lubricity, hydrophobicity Conductive polymericElectric conductivity materials, e.g.. polyvinylpyridine, polyacetylene,polypyrrole) Carbohydrates Alginic acid Lubricity, hydrophilicityCellulose Lubricity, hydrophilicity, bio-degradable glucos sourceChitosan Positively charged surface, hydrophilicity, hemostatsisGlycogen Hydrophilicity, biodegradable glucose source HeparinAntithrombogenicity, hydrophilicity, cell and growth factor attachment,protein affinity Hyaluronic acid Lubricity, negatively charged surfacePectin Lubricity, hydrophilicity Mono-, di-saccharides HydrophilicityDextran sulfate Chromatography media, hydrophilicity Proteins AntibodiesAntigen binding, immunoassay Antithrombotic agents Antithrombogenicsurface (e.g. antithrombin III) Albumin Nonthrombogenic surfaceAttachment Cell attachment proteins/peptides (e.g. collagen) EnzymesCatalytic surface Extracellular matrix Cell attachment and growthproteins/peptides Growth factors, Cell growth proteins/peptides HirudinAntithrombogenic surface Thrombolytic proteins Thrombolytic activity(e.g., streptokinase, plasmin, urokinase) Lipids Fatty acidsHydrophobicity, biocompatibility Mono-, di- and Hydrophobicity,lubricity, bio-degradable fatty triglycerides acid source PhospholipidsHydrophobicity, lubricity, bio-degradable fatty acid sourceProstaglandins/ Nonthrombogenic surface/immobilized leukotrienesmessenger Nucleic Acids DNA Substrate for nucleases/affinity binding,genomic assay RNA Substrate for nucleases/affinity binding, genomicassay Nucleosides, nucleotides Source of purines, pyrimidines, enzymecofactor Drugs/vitamins/cofactors Enzyme cofactors Immobilized enzymeHeme compounds Globin bindings/surface oxygenation Drugs Drug activityNonpolymeric Materials Dyes (e.g., azo dyestuffs) Coloring agentFluorescent compounds Fuorescence (e.g., fluorescein)

The thermally responsive nanofibers of the present invention can be usedin a wide variety of applications including: filters, scaffolds fortissue engineering, protective clothing, reinforcement of compositematerials, and sensor technologies.

Medical articles that can be fabricated from or coated or treated withthe thermally responsive nanofibers of the present invention caninclude, but are not limited to, the following: catheters includingurinary catheters and vascular catheters (e.g., peripheal and centralvascular catheters), wound drainage tubes, arterial grafts, soft tissuepatches, gloves, shunts, stents, tracheal catheters, wound dressings,sutures, guide wires and prosthetic devices (e.g., heart valves andLVADs). Vascular catheters which can be prepared according to thepresent invention include, but are not limited to, single and multiplelumen central venous catheters, peripherally inserted central venouscatheters, emergency infusion catheters, percutaneous sheath introducersystems, thermodilution catheters, including the hubs and ports of suchvascular catheters, leads to electronic devices such as pacemakers,defibrillators, artificial hearts, and implanted biosensors.

Additional articles that can be fabricated from or have a surface thatcan be coated or treated with the thermally responsive nanofibers of thepresent invention can inlude, but are not limited to, the following:slides, multi-well plates, Petri dishes, tissue culture slides, tissueculture plates, tissue culture flasks, cell culture devices, or columnsupports and/or chromatography media.

In another embodiment, the thermally responsive nanofibers of thepresent invention can be applied to a microscope slide or “chip” forbiomolecule immobilization.

In yet another embodiment, the thermally responsive nanofibers of thepresent invention can be applied to a surface of as cell culture deviceto provide as thermally responsive surface

Various types of mammalian cells have been seeded on tissue culturepolystyrene (TCPS) coated with poly-isopropylacrylamide (PIPAAm), Thecells adhered, proliferated and differentiated in the same manner asuncoated TCPS. With the cells on hare TCPS, digestive trypsin treatmentis carried out to dissolve the extracellular matrix and to chelate andremove Ca ions to release the cells, which in the process lose theircell surface receptors, gap junctions and underlying extracellularmatrix. Another alternative for cell release is the use of cellscrapers, the mechanical use of which generates irregularly shapedtissue fragments. With thermo-responsive polymer coated dishes the cellsare detached in a non invasive fashion only by reducing the culturetemperature from 20°-32° C. at a temperature at which the polymerhydrates. In contrast to enzymatic digestion, both adhesive proteins andcell-cell junctions between the confluent cells are preserved, enablinggeneration of a three dimensional functional tissue that lacks anyscaffold.

Cell sheet engineering is a unique technique that has arisen from theuse of thermo-responsive polymer as a cell culture substrate. At 37° C.,PIPAAm becomes hydrophobic, promoting protein adsorption and therebycell adhesion. By lowering the temperature to 20°-32° C., cells can bereleased from the underlying substrate. The change from hydrophobic tohydrophilic character over this transition results in the release ofproteins and adherent cells from the culture substrate. Through thistechnique, cell-cell contacts, gap junctions and surface receptors aremaintained, as well as the underlying extracellular matrix (ECM). Theintact ECM serves as glue to layer cell sheets to form homogenous tissuegrafts for example highly pulsatile cardiac tissue grafts orheterogeneous tissue grafts by layering sheets from various differentcell types, for example endothelial cells and hepatocytes. The cellsheets thus generated have been highly applicable to animal transplantstudies. Transplant experiments have been do compare the response ofdissociated cells versus cell sheet injections. Dissociatedcardiomyocytes equivalent to four cell sheets were injected into leftsubcutaneous dorsal tissue and four cell sheet layers obtained from lowtemperature lift off mediated by thermo-responsive polymer, weretransplanted into the right subcutaneous tissue. The isolated cellsformed a lump under the skin while the sheet transplanted site remainedsmooth. One week after the transplant, the respective sites were openedand cross sectional views of the right side indicated a flat squarecardiac graft with no visible necrosis and connexin 43 (a gap junctionmarker) staining revealed the presence of numerous gap junctions.

The left side showed cell dense graft surface zones with centralcell-void area and only a few depositions were seen when stained forconnexin 43. The grafting of PIPAAm on tissue culture polystyrene andits success with the culture and harvest of various cell types has ledto the development of commercially available tissue culture polystyrenedishes by Cell Seed Inc. (Tokyo, Japan). The use of these surfacescompletely abandons the use of trypsin when collecting cells asdetachment is achieved by lowering the culture temperature. Thiseliminates the use of laborious pipetting, saving on both labor, timeand cell/tissue damage.

The culture surfaces can also be functionalized by co-polymerization ofPIPAAm with its carboxylate derivatized analog,2-carboxyisopropylacrylamide (CIPAAm). Insulin was immobilized onculture surfaces by standard amide bond formation with the CIPAAmcarboxylate group. The surfaces with immobilized insulin showed anincrase in proliferation of bovine carotid artery endothelial (BAEC)cells even without the addition of serum. Similarly, the carboxyl groupson CIPPAm sequences can be used to immobilize cell adhesive sequencessuch as RGDS which promotes BAEC cell adhesion and proliferation withoutthe addition of fetal bovine serum in the culture medium. Thus, theculture of cells and their low temperature lift off obviates the need ofusing serum which has both cost and safety (prions and bovine spongiformencephalopathy) concerns regarding its use. These surfaces would beuseful for serum free culture of cells and cell sheets which can then beused in various tissue engineering and transplant applications.

The spontaneous cell sheet generation from PIPAAm-grafted TCPS is arelatively slow process, occurring gradually from the sheet peripherytoward the interior. Thus, significant incubation time is required tolift up the intact, viable cell sheet completely. Rapid recovery of cellsheets is considered important to maintain biological function andviability of recovered cell sheets, as well as for practical assembly oftissue structures. The rate limiting step to cell recovery is thehydration of hydrophobized PIPAAm segments interacting with the cellsheet, incorporation of a highly water permeable substrate to interfaceis desirable between cell sheets and the thermo-responsive surfaces.Several approaches have been tried in this regard to make the detachmentof cell sheets a faster process. It has been shown that placinghydrophilically-modified PVDF membranes on confluent Madin-Darby caninekidney (MDCK) cells incubated at 20° C. for one hour helps in the easylift of cells. Another set of experiments has utilized porous membranes(PET) grafted with PIPAAm. As mentioned earlier, on PIPAAm-grafted TCPSdishes, water required to hydrate PIPAAm at a lower temperature canreadily penetrate the culture matrix from only the periphery of eachcell to the interface between the cell and grafted PIPAAm chains. Onporous membranes, water hydration of PIPAAm is supplied through poresunderneath adherent cells, as well as from the periphery of each cell.Ready, rapid access of bulk water to PIPAAm grafts through pores beneathattached cells should accelerate single cell and cell sheet detachment.The pore size of a membrane is an important factor in determining thecell adhesion and growth. In general, cells do not grow on surfaceswhich have a pore size greater than their pseudopodium. On membraneswith pore size greater than 5 μm the fibroblast adhesion and suppressionwas found to be greatly reduced.

Nanofibers produced via the process of eleetrospinning may haveunprecedented porosity (>70%), a high surface to volume ratio, and awide range of pore diameter distribution and high interconnectivity, allphysical properties ideal for promoting cell attachment and growth.Furthermore, the nanotopography of electrospun nanofibers closelyresembles the nanofibrillar and nanoporous 3D geometry of the ECM andbasement membrane. The higher surface area allows for a higherpercentage of cellular attachment as well as for multiple focal adhesionpoints on different fibers due to nano-sized fiber diameters. Becausethe diameters of nanofibers are orders of magnitude smaller than thesize of the cells, cells are able to organize, spread or attach toadsorbed proteins at multiple focal points.

Electrospun nanofibers are capable of supporting a wide variety of celltypes. Human umbilical cord endothelial cells attached and proliferatedbetter when seeded onto 50:50 poly (L-lactic acid-co-ε-caprolactone)(PLCL) fibers with a diameter of 300 nm compared to 7 μm microfibers.Cells attached to microfibers were round in shape and non-proliferative,whereas on nanofibers, the cells were nicely spread out and anchored onmultiple fibers. Elias and co-workers have reported osteoblast adhesion,proliferation, alkaline phosphatase activity and ECM secretion on carbonnanofibers increased with decreasing fiber diameter in the range of60-200 nm. Nanogrooved surfaces can induce contact guidance of humancorneal epithelial cells, causing them to elongate and align theircytoskeleton along the topological features. Highly porous PLLAscaffolds with nanoscale pores created using a liquid-liquid phaseseparation have been used for the culture of neural stem cells and wereshown to have a positive effect on neurite outgrowth. Recent studiesshow that the growth of NIH 3T3 fibroblasts and normal rat kidney cellson polyamide nanofibrillar surfaces resulted in changes in morphology,actin organization, focal adhesion assembly, fibronectin secretion andrates of cell proliferation that are more representative of fibroblastphenotype in vivo. Breast epithelial cells on the same surface underwentmorphogenesis to form multicellular spheroids unlike the same cellscultured on glass. It has also been shown that the commerciallyavailable polyamide nanofibers provide a better substrate for cellattachment for weakly adherent cell lines, for example PC12, a neuronalcell line. Polyamide electrospun nanofibers have also been shown tosupport the attachment and proliferation of mouse embryonic stem cells(ES-D3). These cells differentiated into neurons, oligodendrocytes andastrocytes based upon the culture media selected. Fetal bovinechondrocytes seeded on nanofibers poly (ε-caprolactone) (PCL) scaffoldswere able to maintain the chondrocytic phenotype during three weeks ofculture, specifically upregulating collagen type IIB expression,indicative of mature chondrocyte phenotype. These studies demonstratethat nanofiber scaffold are not only cytocompatible but can also be usedto stimulate and encourage cell proliferation and phenotypic behavior.

To induce specific biological responses from the attached cells, thenanofibers may also be functionalized using bioactive molecules.Functionalization is typically carried out by either conjugating themolecules to the surface of the nanofibers or by incorporating thebioactive molecules in the spinning solution. Polyacrylic acid (PAA)grafted onto poly (ε-caprolactone-coethyl ethylene phosphate) (PCLEEP)allows for the conjugation of galactose ligand, which mediateshepatocytes attachment. Hepatocytes cultured on these PCLEEPfunctionalized nanofiber scaffolds formed 20-100 μm spheroid aggregatesthat engulfed the nanofibers. To underscore the importance of culturesubstrate, others have shown that aminated nanofiber meshes supported ahigher degree of cell adhesion and proliferation of hematopoieticstem/progenitor cells compared to aminated films. Similarly conjugationof bone morphogenetic protein-2 (BMP-2) on chitosan nanofibers resultedin better proliferation, alkaline phosphatase activity and calciumdeposition of osteoblastic cells.

In sum, the nanoscale nature of the electrospun polymeric nanofibersmimics the natural ECM, ECM—like properties of the nanofibers can beused to stimulate and encourage cell proliferation and differentiation.Moreover, the cells are able to maintain their in vivo like morphologyand function. Thus, the combination of fiber composition, morphology,alignment and the capacity to incorporate bioactive molecules or growthfactors helps recreate the functions of native ECM.

The invention will be further described with reference to the followingnonlimiting examples. It will be apparent to those skilled in the artthat many changes can be made in the embodiments described withoutdeparting from the scope of the present invention. Thus the scope of thepresent invention should not be limited to the embodiments described inthis application, but only by embodiments described by the language ofthe claims and the equivalents of those embodiments. Unless otherwiseindicated, all percentages are by weight.

EXAMPLES Example 1 Electrospinning of PCL and PS Nanofibers

Poly (ε-caprolactone) (PCL), with an average molecular weight of 80 kDaand Polystyrene (350,000 Da) were purchased from Aldrich chemicals(Milwaukee, Wis.). 0.14 g/ml solutions were prepared by dissolving 14 gof PCL or PS in 100 ml of organic solvent mixture (1:1) composed oftetrahydrofuran (Fisher Scientific) and N,N-dimethylformamide (AlfaAesar, Ward. Hill, Mass.) and mixing it well by shaking the mixture for24 h at room temperature. The polymer solution was placed in a plasticsyringe fitted with a 27 G blunt needle (Strategic Applications, Inc.,Libertyville, Ill.). A syringe pump (KD Scientific, USA) was used tofeed the polymer solution into the needle up. Nanofiber meshes werefabricated by electrospinning using a high voltage power supply (GammaHigh Voltage Research, USA), The nanofibers were collected onto groundedaluminum foil located at a fixed distance from the needle tip. Themeshes were then removed, placed in a vacuum chamber for at least 48 hto remove organic solvent residue and then stored in a dessicator. Thenanofibers were evaluated with a microscope (Olympus BX 60).

Parameters that significantly influence the diameter, consistency anduniformity of the electrospun PCL and PS fibers were polymerconcentration, applied voltage, solution feeding rate andneedle-collector distance. These parameters were optimized untilunbeaded and uniform fibers were spun continuously without needleclogging, Three polymer concentrations (0.10 g/ml, 0.12 g/ml, and 0.14g/ml), two voltages (17 kv, 20 kv) and three needle-collector distances(8 cm, 12 cm, 15 cm) were investigated to obtaim non-defect nanofibers.The optimized conditions are shown in Table 1.

TABLE 1 Electrospinning parameters Polymer concentration 0.14 g/mlApplied voltage 20 kv Flow rate 0.02 ml/min Needle-collector distance 12cm

FIG. 1. shows the typical SEM image of PCL nanofibers. The average fiberdiameter of nanofibers is, 453±146 nm. Highly porous structure wasobserved in the formulation tested. The porosity measured by a liquiddisplacement method was 0.90

Example 2 Coating of Nanofiber Meshes with (PIPAAm)

Various coating approaches were employed to obtain a thin coating of thethermo-responsive polymer on different culture substrates. Thesubstrates included TCPS, Thermanox coverslips (Nunc), commerciallyavailable nanofiber meshes (Surmodics Inc., Corning Inc.) and in-housePCL nanofibers. The Thermanox coverslips and nanofiber inserts were dipcoated in an IPA (isopropyl alcohol) solution of 20 mg/ml PIPAAm(polyisopropylacrylamide Aldrich chemicals, Mw+20-25KDa WI) and 0.8mg/ml TriLite (tris[2-hydroxy-3-(4-benzoylphenoxy)propl]isocyanurate.The pieces were dip coated by immersing in the coating solution for 10seconds and then extracted at a speed of 0.5 cm/sec. The meshes were airdried and then UV illuminated (300-400 nm, Harland Medical UVM400, MN)for 5 minutes. Various dipping speeds, concentrations, number of dipsand immerse times were tried. The efficacy of the coated surfaces wastested by the attachment and detachment behavior of the BAEC cells(Lonza Biosciences, NJ, USA) at 37° C. and 20° C. respectively. Althoughthe above mentioned conditions worked well for cell attachment anddetachment, this dipping method could not coat the tissue cultureformatted surfaces (for example, multi well dishes or 100 mm dishes).Therefore, another approach was tried where the multi well dishes,nanofiber inserts, commercially available nanofiber 96 well and 100 mmdishes were first coated with 0.8 mg/ml solution of TriLite. The TriLitesolution was immediately withdrawn and LTV illuminated for 30 seconds.PIPAAm solution (20 mg/ml in IPA) was then added to the wells,immediately withdrawn and UV illuminated for 2.0 minutes. The treatedsurfaces were rinsed with IPA and tissue culture grade sterile waterbefore plating the cells.

Example 3 Smart Polymer Nanofibers

Four formulations containing 1.0 wt % TriLite were prepared tosynthesize smart polymer photoreactive nanofibers. These formulationswere PS in (DMF/THE), PCL in (DMF/THF), PIPAAm in (IPA/DMF),PIPAAm-co-PEG (1%) in water. The nanofibers were fabricated by theelectrospinning process of Example 2. The parameters such as, polymerconcentration, solvent ratio, applied voltage and needle-collectordistance, were optimized until unleaded and uniform fibers with anaverage diameter under 500 nm can be spun continuously without needleclogging. The optimized conditions are shown in Table 2. After drying,all the nanofibers except PS and PCL were illuminated for 5 minutesunder a UV lamp (Harland Medical UVM400, Eden Prairie, Minn.). Thenanofibers were evaluated under a microscope. PEG-PIPAAm was synthesizedby free radical copolymerization of N-isopropylacrylamide (Aldrich) withpoly (ethyleneglycol) methyl ether methacrylate (Mw 2,000, Aldrich) inwater using ammonium persulfate (Sigma) as initiator andN,N,N′,N′-tetramethylethylenediamine (Aldrich) as a catalyst.

TABLE 2 Feeding Needle- Polymer Applied Rate Collector Polymer SolventConcentration Voltage ml/min Distance PS THF/DMF 14% 20 kv .02 12 cm PCLTHF/DMF 14% 20 kv .02 12 cm PIPAAm IPA/DMF 25% 16 kv 0.1  6 cm PEG-water 5% 16 kv 0.2  6 cm PIPAAm

Example 4 Surface Characterization and Screening of PIPAAm CoatedNanofibers

As the coated nanofiber meshes were completed they were examined forsurface topography, protein adsorption, and contact angle. Initiallythey were screened in house for uniformity by microscopic examination(looking for changes in pore size, and obvious delamination or uncoatedareas) and for contact angles. Comparison between bulk PIPAAm, thecoated and uncoated nanofiber mesh, and the hydrophobic polystyrene corewill provide evidence for surface changes. Microscopic examination ofthe coated nanofibers showed no obvious delamination or changes in themorphology compared to the uncoated nanofibers.

Coatings which passed initial screening were assessed for proteinadsorption. PIPAAm surfaces at 37° C. should adsorb considerably moreprotein than at 25° C. because of the phase transition. Coated anduncoated TCPS coverslips, incubated in 1×PBS buffer at 37° C. and 4° C.for two hours, then quickly removed and placed in a solution of 1 mg/mlBSA for 6 hours at 37° C. and 4° C. This time period, should be enoughfor protein adsorption to occur. Following the BSA incubation, thepieces were rinsed three times with 1×PBS and placed in HRP-labeledanti-BSA antibody (Sigma) for 30 minutes, followed by a standard rinseand HRP colorimetric assay. Pieces were considered coated with thethermo-responsive polymer if the difference in protein adsorptionbetween 37 ° C. and 25° C. incubated coated pieces exceeds one standarddeviation and differs significantly from that of uncoated pieces.

Alternatively, we also adsorbed a cell adhesive protein, Fibronectin(FN). Bovine plasma FN (Biomedical Technology Inc, MA) was adsorbed ontothe nanofibrillar surfaces by incubation of 10 μg/ml FN in PBS solutionat 37° C. and 25° C. for 6 hours. The coated pieces were then vigorouslywashed with PBS for five times. They were blocked with 0.1% bovine serumalbumin (BSA) in PBS for one hour and reacted with 2.0 mg/ml rabbitpolyclonal anti bovine FN antibody (Biogenesis, Inc, UK) at a 1:200dilution (final concentration, 10 μg/ml) for 2 hours at 37° C. and 25°C. respectively. Following five washes with PBS, containing 0.1% BSA,they were incubated for an additional one hour with anti rabbit IgG-HRPantibody (Chemicon International, CA) with a 1:1000 dilution (finalconcentration 15 μg/ml) and incubated with HRP substrate for 10 minutes.The color development was quenched with 1.0N H2S04 and absorbancemeasurements were taken at 450 nm with a spectrophotometer (SpectramaxM2).

A ten fold difference in protein adsorption was seen on PIPAAm coatedsurfaces incubated at 37° C. and 25° C. respectively. Surfaces thatshowed a difference in protein adsorption at 37° C. and 25° C. werefurther evaluated for their cell attachment and detachment profile byplating different cell lines.

Example 5 Cultured Bovine and Human Cells

Bovine Aortic Endothelial Cells (BAEC) and T47-D cells were pre-culturedin 75 cm² flasks in DMEM-F12+10% PBS. The cells were trypsinized andplated on PIPAAm coated nanofibrillar and TCPS surfaces. The cells werealso plated on commercially available PIPAAm coated TCPS surfaces (CellSeed Inc). Both cell lines were plated at a density of 100,000cells/well in a 6-well PIPAAm coated dishes. Bare TCPS and Cell Seedsurfaces were used as control surfaces and similar numbers of cells wereplated on them. The cells were cultured for a period of 48 hours in ahumidified atmosphere with 5% C02 at 37° C. Both the cell lines attachedwell to the coated surfaces which indicate that the coating is thinenough for the cells to attach. Forty eight hours later, the cells weremoved to room temperature. The BAEC cells plated on PIPAAm coatednanofibers, TCPS and Cell seed surfaces started to lift up in about15-20 minutes. Approximately, after about 35 minutes complete cellsheets lifted up (FIG. 4). The results were more dramatic with T47-Dcells. After 25 minutes incubation at room temperature, the cells begunto sheet off from the PIPAAm/TriLite coated nanofibrillar and TCPSsurfaces while the cells plated on Cell Seed surfaces failed to lift upeven after 120 minutes of incubation at room temperature. It wasobserved that on Cell Seed surfaces, there was no cell detachment while50-70% of the cells lifted up from PIPAAm/TriLite coated surfaces inabout half the time (FIG. 5).

Example 6 Cultured Human Epithelial Cells

It has been shown that cells growing on nanofibrillar surfaces form morein vivo like morphologies. These surfaces are also permissive forepithelial cells to undergo morphogenesis. We have shown that ourcoating on smart polymer surfaces does not interfere with thenanofibrillar properties of the matrix and cells still undergomorphogenesis or form more in vivo like structures in addition to beingdetached by mere temperature reduction.

For morphogenesis studies, T47-D breast epithelial cells were culturedon nanofibrillar and flat surfaces coated with PIPAAm/TriLite. Thecontrols were bare nanofibers and TCPS. The cells were cultured inDMEM+10% fetal bovine serum (FBS) in an atmosphere of 5% CO₂ 95% air at37° C. This particular cell line has been selected as it has shown todemonstrate tubular and spheroidal structures under conditions thatpromote three dimensional interactions with collagen or matrigel. After,10 days in culture, cells were fixed with 4% paraformaldehyde andincubated with Phalloidin Alexa Fluor 594 (1:500, Molecular Probes, OR)for 30 minutes at room temperature. The cells were rinsed three timeswith PBS and observed under an inverted fluorescent microscope (ZeissAxiovert 200M). Phalloidin binds to filamentous actin (F-actin) andprovides visualization of cytoskeletal organization of the cells. After5 days in culture, a mixed population of spheroids and tubular cells wasobserved on nanofibers. By day 8, multicellular spheroids were dominantalthough some tubules still persisted. In contrast, the growth of T47-Dcells on flat surfaces showed a monolayer with a group of stress fibers.We have shown that our coating on the nanofiber surface with thethermo-responsive polymer does not affect the nanofibrillar topology andhence morphogensis of T47-D cells or more in vivo like cells can beobtained on these surfaces by mere reduction of temperature. To showthat detached cells recover quickly on fresh surfaces and still retaintheir morphology after temperature reduction, the second set of cellswas grown to confluency for about 5-10 days at 37° C. The cells werethen moved to room temperature for about 15-40 minutes. The detachedcell sheet was gently removed with the help of a 10.0 ml pipette to afresh tissue culture surface. The cells were allowed to settle down andwere then fixed with 4% paraformaldehyde after 30 minutes incubation at37° C. Replated cells were stained with phalloidin F-actin to show thatthe advantage of growing cells on thermo-responsive nanofibrillarSurfaces as opposed to flat thermo-responsive surfaces is the ability toachieve and retain in vivo like morphology (D).

FIG. 7A shows the T47-D cells cultured on PIPAAm/TriLite coatednanofibers for a period of 10 days and stained with Phalloidin F-actin.Note the presence of multicellular spheroids and the peripheralorganization of actin filaments. A magnified image (400 μm) of thespheroid of FIG. 7B shows the lumen extending through the spheroid.T47-D cells cultured on PIPAAm/TriLite coated TCPS for period of 10 dayswere also fixed and stained for phalloidin F-actin. Note the spread outmorphology and organization of stress fibers in the cells of FIG. 7C.T47-D cells lifted up through temperature reduction were replated onfresh nanofibers. FIG. 71) shows that replaced T47-1) cells maintaintheir tubular and spheroidal morphology and peripheral organization ofactin (200 μm).

Replated cell sheets were also analyzed for conexxin 43 expression whichis considered to be a major component of gap junctional channel. BAECcells were plated at a density of 50,000 cells/22 mm well. The cellswere cultured until confluency and then titled up by moving the dish to20° C. for 15 minutes. The sheets were transferred onto freshnanofibrillar surfaces with the help of a 10 ml pipette and the curledup edges were uncurled by adding a drop of medium onto the sheet. Thesheet was then transferred to the 5% CO2 humidified incubator at 37° C.and were allowed to attach. Thirty minutes later the cells were fixedwith 4%, paraformaldehyde and stained with 1:1000 dilution of anticonnexin 43 (Sigma). Staining for Connexin 43 showed diffused expressionof connexin 43 through out the entire sheet suggesting the presence ofintact gap junctions (FIG. 8).

1.-52. (canceled)
 53. A cell culture article comprising a surface and a coating composition in contact with the surface, the coating composition comprising: (a) a thermally responsive polymer, and (b) a monomeric or polymeric crosslinking agent having at least two latent photoreactive groups capable of forming covalent bonds when the coating composition is subjected to electromagnetic energy, thereby coupling the thermally responsive polymer to the surface of the cell culture article in a manner in which at least some of the latent photoreactive groups remain in an inactive state.
 54. The article according to claim 53 wherein the latent photoreactive groups are capable of forming a covalent bond with the surface.
 55. The article according to claim 54 wherein the covalent bond is formed by carbon or nitrogen bond insertion, hydrogen abstraction followed by radical recombination, or dimerization.
 56. The article according to claim 53 wherein the latent photoreactive groups are aryl ketones.
 57. The article according to claim 56 wherein the aryl ketones are selected from acetophenones, benzophenones, anthraquinones, anthrones, and anthrone-like heterocycles.
 58. The article according to claim 53 wherein the crosslinking agent is a compound having a formula selected from (a) to (g): (a) L-(D-T-C(R¹)(XP)CHR²GR³C(═O)R⁴)_(m) wherein L is a linking group; D is O, S, SO, SO₂, NR⁵ or CR⁶R⁷; T is (—CH₂—)_(x), (—CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂CH₂—O—)_(x) or forms a bond; R¹ is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; X is O, S, or NR⁸R⁹; P is a hydrogen atom or a protecting group, with the proviso that P is absent when X is NR⁸R⁹; R² is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; R³ and R⁴ are each independently an alkyl, aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group or optionally, R³ and R⁴ can be tethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s), (—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂—)_(s), (—CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)NR(—CH₂—)_(s); R⁵ and R¹⁰ are a hydrogen atom or an alkyl, aryl or arylalkyl group; R⁶ and R⁷ are each independently a hydrogen atom, an alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl group; R⁸ and R⁹ are each independently a hydrogen atom, an alkyl, aryl, or arylalkyl group; R is a hydrogen atom, an alkyl or an aryl group; q is an integer from 1 to about 7; r is an integer from 0 to about 3; s is an integer from 0 to about 3; m is an integer from 2 to about 10; t is an integer from 1 to about 10; and x is an integer from 1 to about 500; (b) L-(T-C(R¹)(XP)CHR²GR³C(═O)R⁴)_(m) wherein L is a linking group; T is (—CH₂—)_(x), (—CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂CH₂—O—)_(x) or forms a bond; R¹ is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; X is O, S, or NR⁸R⁹; P is a hydrogen atom or a protecting group, with the proviso that P is absent when X is NR⁸R⁹; R² is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; R³ and R⁴ are each independently an alkyl, aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group, or optionally, R³ and R⁴ can be tethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s), (—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂—)_(s), (—CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)NR(—CH₂—)_(s); R¹⁰ is a hydrogen atom or an alkyl, aryl or arylalkyl group; R⁸ and R⁹ are each independently a hydrogen atom, an alkyl, aryl, or arylalkyl group; R is a hydrogen atom, an alkyl or aryl group; q is an integer from 1 to about 7; r is an integer from 0 to about 3; s is an integer from 0 to about 3; m is an integer from 2 to about 10; t is an integer from 1 to about 10; and x is an integer from 1 to about 500; (c) L-(GTZR³C(═O)R⁴)_(m) wherein L is a linking group; T is (—CH₂—)_(x), (—CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂CH₂—O—)_(x) or forms a bond; G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; Z is C═O, COO, or CONH when T is (—CH₂—)_(x); R³ and R⁴ are each independently an alkyl, aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group, or optionally, R³ and R⁴ can be tethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s), (—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂—)_(s), (—CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)NR(—CH₂—)_(s); R¹⁰ is a hydrogen atom or an alkyl, aryl or arylalkyl group; R is a hydrogen atom or an alkyl or aryl group; q is an integer from 1 to about 7; r is an integer from 0 to about 3; s is an integer from 0 to about 3; m is an integer from 2 to about 10; t is an integer from 1 to about 10; and x is an integer from 1 to about 500; (d) L-(TGQR³C(═O)R⁴)_(m) wherein L is a linking group; T is (—CH₂—)_(x), (—CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂CH₂—O—)_(x) or forms a bond; G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; Q is (—CH₂—)_(p), (—CH₂CH₂—O—)_(p), (—CH₂CH₂CH₂—O—)_(p) or (—CH₂CH₂CH₂CH₂—O—)_(p); R³ and R⁴ are each independently an alkyl, aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group, or optionally, R³ and R⁴ can be tethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s), (—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂—)_(s), (—CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)NR(—CH₂—)_(s); R¹⁰ is a hydrogen atom or an alkyl, aryl, or an arylalkyl group; R is a hydrogen atom or an alkyl or aryl group; q is an integer from 1 to about 7; r is an integer from 0 to about 3; s is an integer from 0 to about 3; m is an integer from 2 to about 10; p is an integer from 1 to about 10; t is an integer from 1 to about 10; and x is an integer from 1 to about 500; (e) L-(—CH₂—)_(xx)C(R¹)(GR³C(═O)R⁴)_(m) wherein L is a linking group; R¹ is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl, or aryloxyaryl group; each G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; each R³ and R⁴ is independently an alkyl, aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group, or optionally, R³ and R⁴ can be tethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s), (—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂—)_(s), (—CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)NR(—CH₂—)_(s); each R¹⁰ is a hydrogen atom or an alkyl, aryl, or an arylalkyl group; each R is a hydrogen atom or an alkyl or aryl group; each q is an integer from 1 to about 7; each r is an integer from 0 to about 3; each s is an integer from 0 to about 3; m is an integer from 2 to about 10; each t is an integer from 1 to about 10; and xx is an integer from 1 to about 10; (f) L-(C(R¹)(XP)CHR²GR³C(═O)R⁴)_(m) wherein L is a linking group; R¹ is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; X is O, S, or NR⁸R⁹; P is a hydrogen atom or a protecting group, with the proviso that P is absent when X is NR⁸R⁹; R² is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; R³ and R⁴ are each independently an alkyl, aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group, or optionally, R³ and R⁴ can be tethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s), (—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂—)_(s), (—CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)NR(—CH₂—)_(s); R⁸ and R⁹ are each independently a hydrogen atom, an alkyl, aryl, or arylalkyl group; R¹⁰ is a hydrogen atom or an alkyl, aryl, or an arylalkyl group; R is a hydrogen atom, an alkyl or an aryl group; q is an integer from 1 to about 7; r is an integer from 0 to about 3; s is an integer from 0 to about 3; m is an integer from 2 to about 10; and t is an integer from 1 to about 10; and (g) L-(GR³C(═O)R⁴)_(m); wherein L is a linking group; G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; R³ and R⁴ are each independently an alkyl, aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group, or optionally, R³ and R⁴ can be tethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s), (—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂—)_(s), (—CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)NR(—CH₂—)_(s); R¹⁰ is a hydrogen atom or an alkyl, aryl or arylalkyl group; R is a hydrogen atom, an alkyl or an aryl group; q is an integer from 1 to about 7; r is an integer from 0 to about 3; s is an integer from 0 to about 3; m is an integer from 2 to about 10; and t is an integer from 1 to about
 10. 59. The article according to claim 53 wherein the cell culture article is selected from slides, multi-well plates, Petri dishes, tissue culture plates, tissue culture flasks, and coverslips.
 60. A cell culture article having a thermally responsive surface, the cell culture article comprising a thermally responsive polymer covalently coupled to a surface of the cell culture article via a monomeric or polymeric crosslinking agent, wherein the crosslinking agent includes at least two latent photoreactive groups, wherein at least one of the latent photoreactive groups has undergone activation when subjected to a suitable energy source to form a covalent bond with the surface of the cell culture article, and remaining latent photoreactive groups are present at a ground state energy level.
 61. The article according to claim 60 wherein the latent photoreactive groups are capable of forming a covalent bond with the surface.
 62. The article according to claim 61 wherein the covalent bond is formed by carbon or nitrogen bond insertion, hydrogen abstraction followed by radical recombination, or dimerization.
 63. The article according to claim 60 wherein the latent photoreactive groups are aryl ketones.
 64. The article according to claim 60 wherein the crosslinking agent is a compound having a formula selected from (a) to (g): (a) L-(D-T-C(R¹)(XP)CHR²GR³C(═O)R⁴)_(m) wherein L is a linking group; D is O, S, SO, SO₂, NR⁵ or CR⁶R⁷; T is (—CH₂—)_(x), (—CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂CH₂—O—)_(x) or forms a bond; R¹ is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; X is O, S, or NR⁸R⁹; P is a hydrogen atom or a protecting group, with the proviso that P is absent when X is NR⁸R⁹; R² is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; R³ and R⁴ are each independently an alkyl, aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group or optionally, R³ and R⁴ can be tethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s), (—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂—)_(s), (—CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)NR(—CH₂—)_(s); R⁵ and R¹⁹ are a hydrogen atom or an alkyl, aryl or arylalkyl group; R⁶ and R⁷ are each independently a hydrogen atom, an alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl group; R⁸ and R⁹ are each independently a hydrogen atom, an alkyl, aryl, or arylalkyl group; R is a hydrogen atom, an alkyl or an aryl group; q is an integer from 1 to about 7; r is an integer from 0 to about 3; s is an integer from 0 to about 3; m is an integer from 2 to about 10; t is an integer from 1 to about 10; and x is an integer from 1 to about 500; (b) L-(T-C(R¹)(XP)CHR²GR³C(═O)R⁴)_(m) wherein L is a linking group; T is (—CH₂—)_(x), (—CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂CH₂—O—)_(x) or forms a bond; R¹ is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; X is O, S, or NR⁸R⁹; P is a hydrogen atom or a protecting group, with the proviso that P is absent when X is NR⁸R⁹; R² is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; R³ and R⁴ are each independently an alkyl, aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group, or optionally, R³ and R⁴ can be tethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s), (—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂—)_(s), (—CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)NR(—CH₂—)_(s); R¹⁰ is a hydrogen atom or an alkyl, aryl or arylalkyl group; R⁸ and R⁹ are each independently a hydrogen atom, an alkyl, aryl, or arylalkyl group; R is a hydrogen atom, an alkyl or aryl group; q is an integer from 1 to about 7; r is an integer from 0 to about 3; s is an integer from 0 to about 3; m is an integer from 2 to about 10; t is an integer from 1 to about 10; and x is an integer from 1 to about 500; (c) L-(GTZR³C(═O)R⁴)_(m) wherein L is a linking group; T is (—CH₂—)_(x), (—CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂CH₂—O—)_(x) or forms a bond; G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; Z is C═O, COO, or CONH when T is (—CH₂—)_(x); R³ and R⁴ are each independently an alkyl, aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group, or optionally, R³ and R⁴ can be tethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s), (—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂—)_(s), (—CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)NR(—CH₂—)_(s); R¹⁰ is a hydrogen atom or an alkyl, aryl or arylalkyl group; R is a hydrogen atom or an alkyl or aryl group; q is an integer from 1 to about 7; r is an integer from 0 to about 3; s is an integer from 0 to about 3; m is an integer from 2 to about 10; t is an integer from 1 to about 10; and x is an integer from 1 to about 500; (d) L-(TGQR³C(═O)R⁴)_(m) wherein L is a linking group; T is (—CH₂—)_(x), (—CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂—O—)_(x), (—CH₂CH₂CH₂CH₂—O—)_(x) or forms a bond; G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; Q is (—CH₂—)_(p), (—CH₂CH₂—O—)_(p), (—CH₂CH₂CH₂—O—)_(p) or (—CH₂CH₂CH₂CH₂—O—)_(p); R³ and R⁴ are each independently an alkyl, aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group, or optionally, R³ and R⁴ can be tethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s), (—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂—)_(s), (—CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)NR(—CH₂—)_(s); R¹⁰ is a hydrogen atom or an alkyl, aryl, or an arylalkyl group; R is a hydrogen atom or an alkyl or aryl group; q is an integer from 1 to about 7; r is an integer from 0 to about 3; s is an integer from 0 to about 3; m is an integer from 2 to about 10; p is an integer from 1 to about 10; t is an integer from 1 to about 10; and x is an integer from 1 to about 500; (e) L-(—CH₂—)_(xx)C(R¹)(GR³C(═O)R⁴)_(m) wherein L is a linking group; R¹ is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl, or aryloxyaryl group; each G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; each R³ and R⁴ is independently an alkyl, aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group, or optionally, R³ and R⁴ can be tethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s), (—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂—)_(s), (—CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)NR(—CH₂—)_(s); each R¹⁰ is a hydrogen atom or an alkyl, aryl, or an arylalkyl group; each R is a hydrogen atom or an alkyl or aryl group; each q is an integer from 1 to about 7; each r is an integer from 0 to about 3; each s is an integer from 0 to about 3; m is an integer from 2 to about 10; each t is an integer from 1 to about 10; and xx is an integer from 1 to about 10; (f) L-(—C(R¹)(XP)CHR²GR³C(═O)R⁴)_(m) wherein L is a linking group; R¹ is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; X is O, S, or NR⁸R⁹; P is a hydrogen atom or a protecting group, with the proviso that P is absent when X is NR⁸R⁹; R² is a hydrogen atom, an alkyl, alkyloxyalkyl, aryl, aryloxyalkyl or aryloxyaryl group; G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; R³ and R⁴ are each independently an alkyl, aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group, or optionally, R³ and R⁴ can be tethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s), (—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂—)_(s), (—CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)NR(—CH₂—)_(s); R⁸ and R⁹ are each independently a hydrogen atom, an alkyl, aryl, or arylalkyl group; R¹⁰ is a hydrogen atom or an alkyl, aryl, or an arylalkyl group; R is a hydrogen atom, an alkyl or an aryl group; q is an integer from 1 to about 7; r is an integer from 0 to about 3; s is an integer from 0 to about 3; m is an integer from 2 to about 10; and t is an integer from 1 to about 10; and (g) L-(GR³C(═O)R⁴)_(m); wherein L is a linking group; G is O, S, SO, SO₂, NR¹⁰, (CH₂)_(t)—O— or C═O; R³ and R⁴ are each independently an alkyl, aryl, arylalkyl, heteroaryl, or an heteroarylalkyl group, or optionally, R³ and R⁴ can be tethered together via (—CH₂—)_(q), (—CH₂—)_(r)C═O(—CH₂—)_(s), (—CH₂—)_(r)S(—CH₂—)_(s), (—CH₂—)_(r)S═O(—CH₂—)_(s), —(CH₂—)_(r)S(O)₂(—CH₂—)_(s), or (—CH₂—)_(r)NR(—CH₂—)_(s); R¹⁰ is a hydrogen atom or an alkyl, aryl or arylalkyl group; R is a hydrogen atom, an alkyl or an aryl group; q is an integer from 1 to about 7; r is an integer from 0 to about 3; s is an integer from 0 to about 3; m is an integer from 2 to about 10; and t is an integer from 1 to about
 10. 65. The article according to claim 60 wherein the cell culture article is selected from slides, multi-well plates, Petri dishes, tissue culture plates, tissue culture flasks, and coverslips.
 66. The article according to claim 53 wherein the surface of the cell culture article further comprises a biologically active material.
 67. A coating composition for coating a substrate, the coating composition comprising: (a) a thermally responsive polymer, and (b) a monomeric or polymeric crosslinking agent having at least two latent photoreactive groups capable of forming covalent bonds with a surface of the substrate when the coating composition is subjected to electromagnetic energy, thereby coupling the thermally responsive polymer to the surface of the substrate in a manner in which at least some of the latent photoreactive groups remain in an inactive state.
 68. The article according to claim 67 wherein the latent photoreactive groups are capable of forming a covalent bond with the surface.
 69. The article according to claim 68 wherein the covalent bond is formed by carbon or nitrogen bond insertion, hydrogen abstraction followed by radical recombination, or dimerization.
 70. The coating composition according to claim 67 wherein the photoreactive groups are aryl ketones.
 71. The coating composition according to claim 70 wherein the aryl ketone aryl ketones are selected from acetophenones, benzophenones, anthraquinones, anthrones, and anthrone-like heterocycles.
 72. The article according to claim 60 wherein the surface of the cell culture article further comprises a biologically active material. 